Abstract
Background
X-linked retinoschisis is a retinovitreal disorder primarily affecting males, starting in childhood. Over time, patients experience deterioration of vision due to the lack of retinoschisin-1 function. In clinical trials performing intravitreal gene delivery in those affected by this disorder, ocular inflammation was observed, which may have masked efficacy. A subsequent study focusing on analyzing the populations of peripheral blood mononuclear cells and cytokines in adults with this disease reported aberrant dendritic cell numbers and cytokine levels in peripheral blood, indicating that adults with this disease may have an altered baseline immunity. Whether the aberrant peripheral immunity in affected adults was a consequence of advanced eye pathology remained unclear. This study focuses on analyzing the populations of blood lymphocyte subsets in children aged 0 to 7 years with X-linked retinoschisis and age-matched controls using flow cytometry.
Results
The fractions of lymphocyte subsets that were CD16a+/CD56+, namely natural killer cells, were significantly lower in blood samples from children with X-linked retinoschisis. In children with X-linked retinoschisis, the fractions of CD3+/CD4 + T cells were higher, and the fractions of CD3 + CD8 + T cells were lower, despite having the same amounts of total CD3 + T cells within their lymphocyte populations. This resulted in a significantly greater CD4/CD8 ratio in children with X-linked retinoschisis compared to age-matched controls.
Conclusions
Alterations were found in blood lymphocyte compositions of children with X-linked retinoschisis within both innate and adaptive immune axes. Some alterations including an elevation of CD4/CD8 ratio in X-linked retinoschisis mirror those previously found in adult patients with this disease. The fact that these abnormalities were present early in this disease indicates that retinoschisin-1 may play a role in regulating immunity in addition to retinal structure. The findings may have implications for future treatments such as ocular gene delivery.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12865-025-00741-4.
Keywords: X-linked retinoschisis, Retinoschisin, Flow cytometry, CD4/CD8 ratio
Background
X-linked retinoschisis (XLRS) is a vitreoretinal disorder that causes visual impairment primarily in males. This disease is characterized by formation of cysts separating retinal layers, a clinical feature referred to as retinoschisis that begins at a young age. XLRS is caused by mutations in the retinoschisin-1 (RS1) gene on the X chromosome, which encodes for the RS1 protein. Important for retinal structure [1], RS1 proteins form octamers via interactions of cysteine59 - cysteine223 (1) and these proteins are secreted from the cells [2].
The median age of onset of symptoms in patients with XLRS is 4 years (but can start as young as 1 year old), and the median age at diagnosis is 7 years [3]. With visual acuity progressively declining, patients with this disease often have mild visual impairment by 12 years of age, and low vision by 25 years of age [3]. Approximately 13% of patients experience vitreous hemorrhage at a median age of 9.4 years and about 9% experience retinal detachment at a similar age, which contributes to temporary or permanent vision loss in this disease [3]. Gene therapy treatments in animal models with XLRS show promise for treating this disease using adeno-associated viruses (AAVs) as delivery vehicles [4–7], but a cure for patients does not exist.
After the approval of an ocular gene therapy, Luxturna®, by the United States Food and Drug Administration for treating RPE65 (retinal pigment epithelium-specific 65 kDa protein)-associated retinal dystrophies, the pace of gene therapy development has accelerated. However, side effects such as ocular inflammation hamper the development of gene therapies. Even though some degree of inflammation is typical after any intraocular surgery, and after intraocular gene therapy specifically, its severity depends on the delivery route as well as the dose. In ocular gene therapies using viral vectors as delivery vehicles for transgenes, immune response against the viral vectors, DNA, or transgenic protein is a common bottleneck (reviewed in [8]). In a phase I/II clinical trial using AAV2/4-RPE65-RPE65 to treat patients with RPE65-associated Leber Congenital Amaurosis (LCA), transient inflammation was noted in a dose-dependent fashion [9]. In gene therapies for XLRS, typically administered intravitreally due to the fragile nature of the retina, severity of immune reactions was also dose-dependent. Two phase I/II clinical trials testing gene augmentation therapies in XLRS patients both encountered immune reactions that required implementation of prophylactic immunosuppressive strategies during the trials. In one trial, the intravitreal injection of an AAV8 gene delivery vector containing the RS1 gene caused ocular inflammation in adult patients with XLRS treated with the high dose (1 × 1011 − 3 × 1011 vector genomes per eye, vg/eye) [10]. Antibodies against the AAV8 capsid were observed, the levels of which appeared to be related to the severity of side effects [10]. In another open label, phase I/II dose escalation clinical trial using the AAV2tYF-CB-RS1 gene therapy vector to treat XLRS, varying degrees of intraocular inflammation were observed, including cells in anterior chamber, vitreous cells or vitritis, most of which were successfully treated with steroids. Three out of 13 (23%) of the participants in the high dose group (who received 6 × 1011 vg/eye) developed chronic uveitis [11]. These observations prompt the question whether certain subjects experience more severe side effects due to their baseline immunity, since uveitis is a condition that involves activation of T and B cells (reviewed in [12]). In study participants, the duration of topical steroid administration appeared to be associated with the titers of AAV neutralizing antibodies in serum, implicating systemic immunoreactivity in ocular events [11]. Echoing this sentiment, the appearance of anterior chamber inflammation seemed to coincide with the rise of serum anti-AAV antibody titers [10]. Varying levels of neutralizing antibody in response to gene therapy may point to differences in B cell reactivity among the participants. For both trials, prophylactic systemic immunosuppression was initially not applied; it was later required for subsequent patients after examination of early data [10, 11], yielding valuable knowledge for treating XLRS.
To determine the immune baseline of XLRS patients and identify any abnormality that may interfere with gene therapy administration, a follow-up study was performed in adults with XLRS using flow cytometry. Flow cytometry can be used to identify and quantify populations of immune cells by labeling cells in blood or tissues with cell type specific markers. Identification of circulating dendritic cells and their subtypes was of particular interest, since classical myeloid dendritic cells are antigen presenters that trigger the adaptive immune response, and the unconventional plasmacytoid dendritic cells are adept at secreting cytokines in response to viral pathogens (reviewed in [13]). In the presence of pathogens, myeloid dendritic cells migrate to the lymph nodes to present the antigens to naïve T cells. The populations of CD11c + myeloid dendritic cells in the peripheral blood of adults with XLRS were significantly smaller than those in age-matched control subjects, whereas their populations of CD123 + plasmacytoid dendritic cells were elevated [14]. Additionally, abnormal cytokine profiles were noted at baseline including elevated serum interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF)-α levels [14]. These findings imply that the baseline peripheral immunity of patients with XLRS may be altered, but the functional implications of these differences are unclear. The XLRS subjects included in that study ranged from 23 to 72 years of age [14], and the immunity of children with XLRS was not investigated. Therefore, it is unclear if these differences in immune cell populations identified by flow cytometry were caused by worsening ocular pathologies in adult subjects with this disease.
A clinical study was initiated by our group to investigate the baseline immune status of patients with XLRS from 0 to 100 years, for which the recruitment is ongoing. In this current report, initial flow cytometry findings from the cohort aged 0–7 including five children with XLRS and five age-matched control subjects are presented.
Methods
Patient recruitment
The research was conducted in accordance with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Patients with molecularly confirmed XLRS were identified through the pediatric inherited eye disorder clinic. They were offered enrollment in the study and if accepted, asked to give a blood sample. Informed consent was obtained from the parents or guardian and assent from children if applicable. All recruited patients were males due to the X-linked nature of this disease.
Age-matched male control subjects without the diagnosis of a genetic eye disease were recruited from the general pediatric ophthalmology clinic; some control subjects underwent standard of care related surgical procedures and had the option for the blood sample to be obtained while under sedation.
The blood samples obtained from XLRS patients and control subjects were analyzed by the University of Iowa Hospitals and Clinics Pathology Core Laboratory. The tests performed were complete blood counts with differentials and flow cytometry. For complete blood counts with differentials, whole blood was labeled with specific markers and visualized by XN-20 (Sysmex Corporation). Differentials were performed on a Sysmex SP50 slide stainer and imager using an automated software, Cellavision (Sysmex Corporation). For flow cytometry, peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation. Antibody labeling and washes were performed following standard procedures established and validated in the Pathology Core Laboratory at the University of Iowa. A 10-color FACSCanto flow cytometer (BD Biosciences) was used and results were analyzed on FlowJo (BD Biosciences) or CytoPaint Classic immunophenotyping software (Leukobyte). Standard flow cytometry panel plus the CD16 and Dendrite Cell Determination analyses were conducted.
To investigate populations of lymphocytes in PBMCs, whole blood was collected, PBMCs were isolated and stained with antibodies, and flow cytometry was performed. To enrich for lymphocytes in subsequent analysis, flow cytometry data was gated by side scatter and CD45, a marker for cells of the hematopoietic lineage. This gating strategy, hereafter referred to as the lymphocyte gate (Fig. 1a), excludes neutrophils, monocytes and granulocytes from subsequent analysis.
Fig. 1.
Quantification of lymphocyte subsets following flow cytometry using innate immune markers in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45 (a), cell populations within the lymphocyte gate that are CD11c+ (b), CD11c+/CD56- (c), CD11c+/CD56+ (d), CD14+ (e), and CD16a+/CD56+ (f) were quantified. Both fractions (%) and absolute counts (#) of each cell population are shown, and errors represent standard error of the mean. Lymphocyte populations gated by CD16a and CD56 in a 3-year-old XLRS subject and an age-matched control are displayed (g)
Data analysis
Statistical analysis was performed using GraphPad PRISM. Comparison between groups were performed using unpaired t-test.
Antibodies
Antibodies used for flow cytometry are as follows: CD4-FITC (BD Biosciences, #340133), CD25-PE (BD Biosciences, #341010), CD3 PERCYPCY5.5 (BD Biosciences, #340948), CD16-PE CY7 (BD Biosciences, #335806), CD19-APC (BD Biosciences, #340722), CD56-APC R700 (BD Biosciences, #657887), CD8-APC H7 (BD Biosciences, #641409), CD127-BV421 (BD Biosciences, # 562436), CD45-V500 (BD Biosciences, # 647450), CD14-BV605 (BD Biosciences, #564054), CD11C-APC (BD Biosciences, #340544).
Results
Patient characteristics
Subjects included in this report were a part of a larger, ongoing study with a target recruitment of at least 20 subjects with XLRS and 20 controls across different ages. In this report, findings from the youngest cohort including five children with XLRS aged 0–7 years and five age-matched control subjects are presented. The characteristics of these children with XLRS and age-matched controls, including genetic mutations and animo acid changes in those with XLRS, are listed in Table 1. The average ages between the children with and without XLRS were not significantly different (p = 0.7707, unpaired t-test). All recruited subjects were males due to the X-linked nature of this disease.
Table 1.
Subject characteristics of children with XLRS and age-matched controls
| Code | Ages (years) | Genetic Mutation | Amino Acid Change | |
|---|---|---|---|---|
| XLRS | R1 | 2 | c.336G > T | p.Trp112Cys (W112C) |
| R2 | 2 | c.214G > C | p.Glu72Gln (E72K) | |
| R3 | 3 | c.214G > C | p.Glu72Gln (E72K) | |
| R4 | 3 | c.214G > C | p.Glu72Gln (E72K) | |
| R5 | 7 | c.286T > C | p.Trp96Arg (W96R) | |
| Average | 3.4 | |||
| Control | C1 | 0 | NONE | NONE |
| C2 | 3 | NONE | NONE | |
| C3 | 3 | NONE | NONE | |
| C4 | 3 | NONE | NONE | |
| C5 | 6 | NONE | NONE | |
| Average | 3 |
One patient (R1) with XLRS had a mutation that resulted in the amino acid change W112C in RS1, patients R2, R3 and R4 had mutations that caused the E72K change, and R5 harbored the W96R substitution. All these changes are located within the discoidin domain of the RS1 protein [15]. The W112C substitution found in patient R1 had previously been noted in a 52-year-old subject with XLRS, who had a mild impairment in visual acuity and light aversion [16]. In cell culture, RS1 proteins with the W112C change are expressed at levels comparable with wild-type (WT) RS1 proteins. However, the secreted form of the W112C mutant protein, either as monomers or as octomers, was not observed [16]. Patients R2– R4 had the E72K amino acid substitution. In cell culture, RS1 proteins with the E72K mutation showed no octamer formation and were not properly secreted from cells compared to WT RS1 proteins [15]. In patient R5, the change from tryptophan, a non-polar amino acid, to arginine, a polar amino acid, at position 96 (W96R) purportedly introduces a positive charge into the cavity between β2 and β3 strands [17]. In cell culture, this mutant protein also had impaired secretion [18]. Together, the amino acid substitutions reported in this study all fall within the discoidin domain of RS1 and reportedly prevent the mutant RS1 from being secreted.
Blood cell counts
To determine if there are major differences in the populations of blood cells, complete blood counts with differentials were performed. There were no significant differences in absolute blood counts nor percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils between children with XLRS and age-matched controls (Table 2). There were also no significant differences in immature granulocyte number, white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, mean platelet volume, nor RBC distribution width (supplementary Table 1).
Table 2.
Leukocyte counts in peripheral blood in children with or without XLRS
| Controls (absolute count) | XLRS (absolute count) | P value | |
|---|---|---|---|
| Neutrophils |
41.04 ± 4.09% (3054.00 ± 393.78 cells/µL) |
41.50 ± 1.99% (3212.00 ± 423.64 cells/µL) |
0.92 (0.79) |
| Lymphocytes |
47.48 ± 4.70% (3474.00 ± 325.03 cells/µL) |
47.44 ± 2.95% (3668.00 ± 489.50 cells/µL) |
0.99 (0.75) |
| Monocytes |
8.04 ± 1.00% (590.00 ± 71.97 cells/µL) |
6.82 ± 0.80% (514.00 ± 75.74 cells/µL) |
0.37 (0.49) |
| Eosinophils |
2.64 ± 0.43% (194.00 ± 31.56 cells/µL) |
3.52 ± 1.06% (242.00 ± 49.13 cells/µL) |
0.46 (0.43) |
| Basophils |
0.58 ± 0.06% (42.00 ± 3.74 cells/µL) |
0.54 ± 0.15% (38.00 ± 5.83 cells/µL) |
0.81 (0.58) |
Flow cytometric analysis using markers associated with innate immunity
To identify lymphocyte subsets related to innate immunity, cell populations were further gated by CD11c, CD16a, and CD56. CD11c is an integrin that plays a critical role in the adhesion of immune cells to endothelial cells prior to extravasation and immune activation. The population of CD11c + cells includes monocytes, granulocytes, a subset of B cells, dendritic cells, and macrophages. Within the lymphocyte gate, children with XLRS tended to have lower percentages of cells that are CD11c+ (Fig. 1b; Table 3). The trend of decrease in the population of CD11c + cells in children with XLRS mirrored findings made in adult XLRS patients [14]. The population of CD11c + cells were then subdivided and gated by their expression of CD56. The percentage of CD11c+/CD56- cells in children with XLRS was not significantly different compared to that in age-matched controls (p = 0.6128, Fig. 1c; Table 3). The fractions of CD11c+/CD56 + cells, natural killer (NK)-like cells important for expansion of ɣδ T cells [19], are shown (p = 0.2200, Fig. 1d; Table 3).
Table 3.
Lymphocyte subsets in children with or without XLRS
| Markers | % lymphocyte gate in controls (absolute count) ± standard error of the mean | % lymphocyte gate in XLRS (absolute count) ± standard error of the mean | P value |
|---|---|---|---|
| CD11c+ |
11.16 ± 1.99% (400.12 ± 101.86 cells/µL) |
7.87 ± 1.04% (274.10 ± 34.56 cells/µL) |
0.18 (0.28) |
| CD11c+/CD56+ |
6.77 ± 1.96% (240.40 ± 86.69 cells/µL) |
4.06 ± 0.56% (141.13 ± 19.63 cells/µL) |
0.22 (0.30) |
| CD11c+/CD56- |
4.39 ± 0.72% (159.71 ± 32.78 cells/µL) |
3.81 ± 0.83% (132.97 ± 28.94 cells/µL) |
0.61 (0.56) |
| CD16a+ |
13.25 ± 3.30% (467.07 ± 147.73 cells/µL) |
4.52 ± 1.27% (150.75 ± 43.49 cells/µL) |
0.039 (0.074) |
| CD16a+/CD56+ |
10.32 ± 2.74% (358.04 ± 118.27 cells/µL) |
3.37 ± 1.05% (112.63 ± 36.89 cells/µL) |
0.045 (0.083) |
| CD14+ |
18.07 ± 7.12% (652.74 ± 303.04 cells/µL) |
11.40 ± 4.03% (482.94 ± 180.75 cells/µL) |
0.44 (0.64) |
| CD19+ |
15.20 ± 2.22% (544.00 ± 100.92 cells/µL) |
20.80 ± 3.12% (768.60 ± 142.74 cells/µL) |
0.18 (0.23) |
| CD3+ |
72.00 ± 3.33% (2481.60 ± 219.59 cells/µL) |
72.40 ± 2.98% (2665.00 ± 397.29 cells/µL) |
0.93 (0.70) |
| CD3+/CD4+ |
36.60 ± 3.14% (1279.00 ± 176.07 cells/µL) |
46.20 ± 2.04% (1723.80 ± 276.16 cells/µL) |
0.033 (0.21) |
| CD3+/CD8+ |
29.20 ± 2.04% (996.40 ± 72.87 cells/µL) |
20.80 ± 1.28% (778.00 ± 132.95 cells/µL) |
0.0082 (0.19) |
| CD3+/CD4-/CD8- |
6.40 ± 1.12% (212.00 ± 25.02 cells/µL) |
5.40 ± 1.91% (163.40 ± 21.86 cells/µL) |
0.66 (0.18) |
| CD3-/CD56+ |
10.60 ± 2.73% (369.80 ± 120.22 cells/µL) |
5.00 ± 0.71% (176.40 ± 28.15 cells/µL) |
0.082 (0.16) |
| CD4+/CD25+ |
7.80 ± 0.74% (264.20 ± 18.30 cells/µL) |
9.20 ± 0.92% (327.00 ± 44.71 cells/µL) |
0.27 (0.23) |
| CD4+/CD25+/CD127- |
3.20 ± 0.37% (108.00 ± 10.33 cells/µL) |
4.20 ± 0.49% (159.20 ± 30.87 cells/µL) |
0.14 (0.15) |
Then, lymphocyte populations expressing CD14 were quantified. CD14 mediates innate immune activation in response to bacteria [20] as well as viral pathogens [21]. In the latter scenario, it serves as a co-receptor for toll-like receptors 7 and 9 to mediate cytokine release [22]. No significant differences were found (Fig. 1e; Table 3).
Found on NK cells, a subset of monocytes, macrophages, and dendritic cells, CD16a is key to antibody-mediated cytotoxicity [23] as it binds to IgG with a 10-fold higher affinity compared to CD16b [24]. NK cells, which are double positive for CD16a and CD56, account for 10% of lymphocytes in controls, compared to only about 3% in children with XLRS (p = 0.0454, Fig. 1f; Table 3), which is 67% lower in comparison. Cell populations within the lymphocyte gate from a 3-year-old XLRS child (R4) and an age-matched control without XLRS (C3) are shown (Fig. 1g). Children with XLRS had fewer NK cells in their circulation.
Flow cytometric analysis using markers associated with adaptive immunity
To quantify lymphocyte subsets belonging to the adaptive immune system, cell populations in the lymphocyte gate were further gated by cell-specific markers (Fig. 2a). B cell counts, which were identified by CD19 expression, showed a trend of elevation in blood samples from children with XLRS, in which the counts were 41% higher on average (p = 0.2348, Fig. 2b; Table 3). Identified by CD3, T-cells play an important role in defenses against infections. T cells were further subdivided into CD4 + and CD8 + subsets, which are considered helper T cells and cytotoxic T cells, respectively. Of note, neither the fractions nor the absolute numbers of all CD3 + cells within the lymphocyte gate were different between children with and without XLRS (Fig. 2c; Table 3). However, the fraction of CD3+/CD4 + cells, that is the percentage of T cells that are helper T cells, was significantly higher in children with XLRS (p = 0.0333, Fig. 2d; Table 3). Representative data is shown in Fig. 2e. On the other hand, the fractions of CD3+/CD8 + cells, considered cytotoxic T-cells, were significantly lower in children with XLRS (p = 0.0082, Fig. 2f, representative data in Fig. 2g; Table 3). The concomitant elevation in CD3+/CD4 + T-cells by 26% and reduction in CD3+/CD8 + T cells by 29% resulted in a higher CD4/CD8 ratio in children with XLRS (controls: 1.274 ± 0.125; XLRS: 2.234 ± 0.081, p = 0.0002). The elevation of CD4/CD8 ratio in children with XLRS mirrors the findings made in adult XLRS subjects by Mishra et al. [14]. The populations of CD3+/CD4-/CD8- cells were not different between controls and children with XLRS (Fig. 3a; Table 3). Within the CD4 + subset, T regulatory cells (Tregs) help modulate the immune response and immunosuppression. To determine whether the populations of Tregs were altered in XLRS, CD4+/CD25+/CD127 − T cells were analyzed. Foxp3 is the master transcriptional regulator for Treg-specific genes [25], and T cells identified by CD4+/CD25+/CD127low or CD127 − are analogous to classical Tregs identified by CD4+/CD25+/Foxp3+ [26]. Although not statistically significant, both the fractions and the absolute counts of CD4+/CD25+/CD127- cells within the lymphocyte gate showed a trend of elevation in children with XLRS (Fig. 3b; Table 3). Populations of lymphocytes gated by CD4 and CD25 are shown (Fig. 3c).
Fig. 2.
Quantification of lymphocyte subsets following flow cytometry using markers related to adaptive immunity in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45 (a), cell populations within the lymphocyte gate that are CD19+ (b), CD3+ (c), CD3+/CD4+ (d, e), CD3+/CD8+ (f, g) were quantified. Both fractions (%) and absolute counts (#) are shown, and errors represent standard error of the mean. Lymphocyte populations for a 3-year-old control subject (C4) and a 3-year-old patient with XLRS (R3) gated by CD3 and CD4 (e) or CD3 and CD8 (g) are displayed
Fig. 3.
Quantification of lymphocyte subsets following flow cytometry using markers related to adaptive immunity in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45, cell populations within the lymphocyte gate that are CD3+/CD4-/CD8- (a), and CD4+/CD25+/CD127- (b) were quantified. Both fractions (%) and absolute counts (#) are displayed, and errors represent standard error of the mean. Data for a 3-year-old control subject (C4) and a 3-year-old patient with XLRS (R3) is shown (c)
Together, the data suggests that T cells shift toward a CD4 identify in children with XLRS. The total numbers of T cells or lymphocytes were not different. A trend towards having a greater B cell pool was noted in children XLRS.
Together, these findings suggest that children with XLRS also have an altered peripheral immunity, similar to observations made in adults.
Discussion
Limitations
The study is limited by sample size (n = 5 for XLRS and n = 5 for controls) as well as the genetic heterogeneity of the mutations in RS1 among these XLRS patients. Nevertheless, these three mutations all fall within the discoidin domain of RS1. Based on literature, these mutations are all expected to negatively impact the ability of the mutant RS1 protein to be secreted.
Due to the small sample size of this study, cell populations expressing certain innate immune markers such as CD11c or CD14 showed a trend of reduction in our dataset, without being statistically significant. On the other hand, cell populations that were CD19 + or CD4+/CD25+/CD127 − tended to be greater in number in children with XLRS, without reaching statistical significance. These results should be interpreted with caution and additional studies are needed.
Apparent reductions in CD16a + and CD11c + cell populations in children with XLRS
A previous study by Mishra et al. investigated the baseline immune status in adults with XLRS [14]; they found aberrations in levels of CD11c + cells and an elevated CD4/CD8 ratio in adult subjects with XLRS [14]. However, it was unclear whether these abnormalities in their peripheral immune cell populations developed over time as their ocular pathology progressed, or whether these alterations were present early. To address this, we have initiated a similar study in XLRS including subjects from different age groups. In this current study, initial findings in the youngest cohort of patients consisting of five children with XLRS and five age-matched controls aged between 0 and 7 are reported.
In terms of markers commonly used to identify innate immune cells, significant reductions in populations of CD16a+/CD56 + cells were identified. Antibodies against CD16a distinguish NK cells from neutrophils [27], whereas antibodies against the CD16b isoform identify neutrophils [28] and granulocytes [29]. Notably, populations of CD16a+/CD56 + NK cells were reduced by 67% in children with XLRS compared to controls. The reduction in this population may have functional consequences that merits further investigation. It is unknown if a reduction in NK cell numbers in XLRS patients is due to a primary defect in the differentiation and/or proliferation of NK cells, or due to NK exhaustion. In NK exhaustion, typically due to over-stimulation, NK cells become functionally impaired and expression of their surface markers is altered (reviewed in [30]). Sometimes, this is accompanied by a reduction in NK number. In patients with chronic hepatitis C infections, for example, the population of CD56dim NK cells in peripheral blood was 4.9% of lymphocytes compared to 9.0% in controls using flow cytometry [31]. Presumably representing cells capable of responding to antibody-coated immune complexes, the functional consequence of a reduction in NK cells is unknown without further study.
In our study, children with XLRS demonstrate a trend of having fewer CD11c+, CD11c+/CD56-, and CD11c+/CD56 + cells in their lymphocyte fractions. However, these changes were not statistically significant, likely due to the sample size in our study. A reduction in the dendritic cell population could mean impaired antigen presentation and/or cytokine secretion.
A shift towards CD4 + T cell identity and elevation of CD4/CD8 ratio in children with XLRS
Previously, an elevation of CD4/CD8 ratio was reported in adult subjects with XLRS at baseline when compared to that in age-matched controls (XLRS base-line: 3.5 ± 0.9; control: 2.2 ± 0.7; p < 0.001) [14]. This finding opened the line of investigation whether an altered adaptive immunity could have impacted gene delivery in XLRS [14]. Similarly, we found that children with XLRS also had a higher number of CD4 + T cells and a lower number of CD8 + T cells, resulting in an elevated CD4/CD8 ratio compared to age-matched control subjects. The fact that this abnormality is present early in XLRS suggests that it could be a primary feature of the disease, and not a consequence of advanced ocular pathology. CD4/CD8 ratio signals the balance between CD4 helper T cells and CD8 cytotoxic T cells. It is a prognostic marker in many diseases, including human immunodeficiency virus (HIV)-associated immune deficiency. In HIV, there is typically an inversion (decrease) in the CD4/CD8 ratio due to the rapid depletion of CD4 + T cells, signaling the loss of ability to fight opportunistic infections [32]. Conversely, an increase in CD4/CD8 ratio may signal an increased immune competence. In an observational, multi-center, and retrospective study investigating factors contributing to COVID-19 disease severity, patients in the critically-ill group had higher CD4/CD8 ratios compared to those in the non-critically ill group after coronavirus infections [33]. Furthermore, medications including tocilizumab and corticosteroids (methyl-prednisolone or dexamethasone) were more frequently used in the critically-ill group than in the non-critically-ill group (46% vs. 4%, p = 0.0062 and 85% vs. 36%, p = 0.0122, respectively). The interaction between the CD40 receptor on B cells and CD40 ligand (CD40L) from helper T cells promotes the differentiation and isotype switching in B cells (reviewed in [34]). This discovery was precipitated by the lack of CD40L production in T cells from patients with hyper IgM syndrome, who have abnormally low levels of IgG [35]. It is unknown whether increases in numbers of both CD4 + T cells and B cells would result in a positive feedback loop. Taken together, these studies could offer support for the notion that possessing an elevated CD4/CD8 ratio, as is the case in children with XLRS, predisposes the subject to experiencing more severe inflammation when faced with viral infections or when undergoing treatments utilizing viral vectors. However, this view remains a conjecture. These findings could have implications for gene therapy development for this disease. The functional consequences of an altered CD4/CD8 ratio need to be further tested.
In the context of viral vector-based gene delivery, the functional consequences of these immune alterations in XLRS are not known without further study. Changes in populations of peripheral immune cells may imply dysfunction, but not necessarily so. However, our report provides additional evidence that both the innate and adaptive immune systems are abnormal in children with XLRS. In the clinical gene therapy trial, elevation in AAV8 capsid-specific antibodies were observed in adults with XLRS after AAV8-mediated gene therapy, and the antibody levels were not the same among individuals receiving the same dose [10]. In another clinical trial for treating XLRS, 23% of the study participants in the high dose group developed chronic uveitis [11], a condition related to activation of T and B cells (reviewed in [12, 36]), but others in the high dose group did not develop this condition. In our study, children with XLRS showed a trend of having greater populations of B cells in their blood, but the statistical power was limited by the sample size of our study. Determining how immune baseline measurements relate to outcomes would be of particular interest.
The fact that young children with XLRS have alterations in peripheral immune cell populations may offer support for the notion that the lack of RS1 gene function causes aberrations in the peripheral immune compartments. However, this remains speculative, as the function of the RS1 gene outside of the eye is poorly understood. In retinal explants and in Y79 retinoblastoma cell lines, the lack of RS1 protein function causes pathological upregulation of ERK MAPK signaling pathway, along with upregulation of proapoptotic transcription factor BAX2 [37]. Conceivably, the lack of RS1 function could impact proliferation and apoptosis in other systems through regulating MAPK signaling. Interestingly, RS1 gene expression is a prognostic marker for lung adenocarcinoma, and RS1 gene expression was lower in tumors [38]. A previously unknown role of the RS1 gene in immunity may be emerging.
Conclusions
Alterations in immune cell populations belonging to both the innate and adaptive axes were found in peripheral blood collected from children with XLRS compared to age-matched controls. Some of these findings mirror observations made in adults with XLRS. Together, this supports the notion that aberrations in the peripheral immune compartment are a direct consequence of loss of RS1 function and not secondary to the worsening ocular pathologies in this disease. However, to demonstrate that this is a perturbation present since birth, this conclusion would be further supported by samples from newborn children with XLRS, or longitudinal studies.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors would like to acknowledge the study participants and their families. The authors would like to acknowledge funding sources including the Chakraborty Family Foundation (PI: Drack), Knights Templar Eye Foundation Career Starter grant (PI: Hsu), Research to Prevent Blindness, and the Ronald Keech Professorship (Drack).
Author contributions
AVD conceptualized the study. GDV, STS, CS, AD and AVD recruited and consented patients. SIS, YH and GDV interpreted the flow cytometry results. YH also analyzed the data, generated the figures and wrote the manuscript. BL, GDV and CRF assisted in data interpretation and scientific visualization. YH, BL, GDV, STS, CS, SS, CRF, AD and AVD critically revised the manuscript.
Funding
The study was supported by Chakraborty Family Foundation (PI: Drack), and Knights Templar Eye Foundation career starter grant (PI: Hsu). The funders do not have a role in experimental design or data analysis.
Data availability
Data is provided within the manuscript or supplementary information files.
Declarations
Ethics approval and consent to participate
The research was conducted in accordance with Declaration of Helsinki and with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Informed consent was obtained from the parents or guardian. Clinical trial number: not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
Data is provided within the manuscript or supplementary information files.